Flowable tissue matrices

ABSTRACT

Disclosed herein are flowable tissue matrix compositions comprising small pieces of partially or completely decellularized tissue suspended in a gelatinized tissue or gelatin gel comprising partially or completely decellularized tissue or synthetic gelatin. The flowable tissue matrix compositions can contain factors that promote or enhance native cell migration, proliferation, and/or revascularization after implantation into a subject. Also disclosed are methods of making and using the flowable tissue matrix compositions. The compositions can be implanted into a tissue in need of repair, regeneration, healing, treatment, and/or alteration, and can promote or enhance native cell migration, proliferation, and/or revascularization.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/868,588, filed Apr. 23, 2013, which claims priority to U.S.Provisional Application No. 61/637,419, filed Apr. 24, 2012, which isincorporated herein by reference in its entirety.

The present disclosure relates generally to methods of making and usingcompositions comprising flowable tissue matrices.

Acellular tissue matrices of both animal and human origin are used forsoft tissue repair and regeneration. Currently, acellular tissue isoften used in sheet form. Sheets of acellular tissue, however, presentpractical limitations, such as limits on the ability to mold the tissueinto a desired shape to match the structure of an anatomical implantsite. Alternative structures, such as particulate acellular tissue, arelimited by their speed of resorption, degradation, or migration awayfrom the site of implantation. In addition, particulate acellular tissueis often stored freeze-dried, thereby requiring time-consumingrehydration prior to use in the surgical setting.

Accordingly, disclosed herein are flowable tissue matrix compositions,comprising small pieces of partially or completely decellularized tissuesuspended in a gelatinized tissue or gelatin gel. In some embodiments,the volume of gelatinized tissue or gelatin gel is minimized in order toreduce the amount of denatured collagen present in the compositionsand/or to avoid disrupting the migration, proliferation, orrevascularization of an implanted composition. In some embodiments, thesmall pieces of partially or completely decellularized tissue in aflowable tissue matrix composition are selected such that a majority ofthe pieces minimize their surface area to volume ratio, for example byproducing pieces having a ratio of less than about 6 mm²/mm³. In someembodiments, the small pieces of partially or completely decellularizedtissue in a flowable tissue matrix composition are selected such that amajority of the pieces have a surface area to volume ratio less thanabout 6.0, 5.5, 5.0, or 4.5 mm²/mm³ (or any value in between) andgreater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0 mm²/mm³ (or any value in between). As used herein, a “majority”indicates at least about 50% of the small pieces (e.g., at least about50, 55, 60, 65, 70 75, 80, 85, 90, 85, 99, or 99.9%) (or any percentagein between).

In some embodiments, the small pieces of decellularized tissue areselected or processed to have dimensions large enough to avoid rapiddegradation, but small enough to exhibit improved flowable or malleablecharacteristics and to allow for the use of a low or reduced amount ofgelatin or gelatin gel. For example, suitable pieces of decellularizedtissue can have dimensions (i.e., a length, width, and/or height)ranging from about 1.0 mm to about 5.0 mm (e.g., dimensions of about1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mm) (or any value inbetween). For example, the dimensions can range between about 1.0 mm and5.0 mm, or between about 1.5 mm and 4.5 mm, or between about 2.0 mm and4.0 mm. In certain embodiments, flowable tissue matrix compositions haveincreased resistance to degradation, migration, or resorption, ascompared to homogenized acellular tissue, while also retaining theability to flow into and mold to the shape of an implant site. In someembodiments, the small pieces of partially or completely decellularizedtissue have a length, a width, and a height, and wherein each of thelength, width, and height have dimensions ranging from about 1.0 mm toabout 5.0 mm.

In various embodiments, the gelatinized tissue or gelatin gel comprisesa synthetic material or a homogenized acellular or partiallydecellularized tissue in an aqueous solution at a concentration of about0.1-10.0% w/v. In certain embodiments, the gelatin gel is crosslinked.

In various embodiments, the small pieces of partially or completelydecellularized tissue, the gelatinized tissue and/or the gelatin gel arederived from at least one of human, nonhuman primate, pig, cow, horse,goat, sheep, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, andmouse tissue, and/or at least one of bone, skin, dermis, intestine,vascular, urinary bladder, tendon, ligament, muscle, fascia, neurologictissue, vessel, liver, heart, lung, kidney, and cartilage tissue. Insome embodiments, the compositions lack substantially allalpha-galactose moieties. In various embodiments, a flowable tissuematrix composition has a reduced bioburden or substantially lacks allbioburden.

In various embodiments, flowable tissue matrix compositions comprise oneor more viable cells, such as stem cells, and/or at least one additionalfactor, such as an anti-inflammatory agent, an analgesic, a cell growthfactor, an angiogenic factor, a differentiation factor, a cytokine, ahormone, or a chemokine.

In some embodiments, a kit is provided, comprising a flowable tissuematrix composition as disclosed herein and instructions for using thekit. In certain embodiments, the kit is packaged under aseptic orsterilize conditions. In some embodiments, the kit comprises a syringeor other delivery device preloaded with a flowable tissue matrixcomposition in aqueous form and ready for delivery to a surgical site ona patient.

In various embodiments, a method is disclosed for making a flowabletissue matrix composition, comprising selecting a tissue containing anextracellular matrix; partially or completely decellularizing thetissue; processing the decellularized tissue to produce small pieces ofthe tissue (e.g., pieces having dimensions ranging from about 1.0 mm toabout 5.0 mm in length); gelatinizing a partially or completelydecellularized tissue; optionally heating and cooling the gelatinizedtissue to produce a gelatin gel; and combining the small pieces ofdecellularized tissue with the gelatinized tissue or gelatin gel. Insome embodiments, processing the partially or completely decellularizedtissue into small pieces comprises cutting the partially or completelydecellularized tissue into pieces having a length, a width, and aheight, and wherein each dimension is between about 1.0 mm and 5.0 mm.In certain embodiments, gelatinizing the partially or completelydecellularized tissue comprises suspending the partially or completelydecellularized tissue in a solution containing one or more Lewis bases(such as sodium carbonate, sodium citrate, or sodium acetate) andhomogenizing the tissue. In some embodiments, producing a gelatin gelcomprises placing a gelatinized tissue in a hydrating solution, heatingthe tissue, and then allowing the tissue to cool. In some embodiments, agelatin gel is selected from biocompatible synthetic materials that havea viscous consistency. In certain embodiments, the gelatin gel iscross-linked by contacting the tissue with a cross-linking agent, suchas pentagalloyl glucose (PGG), glutaraldehyde, or genipin.

In various embodiments, the small pieces of decellularized tissue arecombined with a minimal volume of gelatinized tissue or gelatin gel inorder to minimize the percentage of gelatinized tissue or gelatin gel inthe composition, as compared to the percentage of small pieces ofdecellularized tissue in the overall composition (measured on amass/volume or volume/volume basis). For example, small pieces ofdecellularized tissue (e.g., pieces having dimensions between about 1.0mm and about 5.0 mm and/or a surface area to volume ratio between about1 mm²/mm³ and 6 mm²/mm³) can be held together in a structurally stablecomposition when up to about 95% (or 90%, 80%, 70%, or 60%, or anypercentage in between) of the composition (w/v or v/v) comprises smallpieces of tissue, with the remaining approximately 5% (or 10%, 20%, 30%,or 40%, or any percentage in between) comprising gelatin or gelatin gel.In contrast, larger pieces of decellularized tissue may requireadditional gelatin or gelatin gel in order to adhere (e.g., to “glue”)the pieces together into a structurally stable composition that will notmigrate (e.g., break apart) into separate and disparate pieces ofdecellularized tissue after implantation (e.g., potentially needing upto 50% or more gelatin or gelatin gel).

In some embodiments, the dimensions of the small pieces ofdecellularized tissue are selected such that a majority of the piecesminimize their surface area to volume ratio, for example, by having aratio of less than about 6 mm²/mm³. In some embodiments, the smallpieces of partially or completely decellularized tissue in a flowabletissue matrix composition are selected such that a majority of thepieces have a surface area to volume ratio of less than about 6.0, 5.5,5.0, or 4.5 mm²/mm³ (or any value in between) and greater than about1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm²/mm³ (or anyvalue in between). As used herein, a “majority” indicates at least about50% of the small pieces (e.g., at least about 50, 55, 60, 65, 70 75, 80,85, 90, 85, 99, or 99.9%) (or any percentage in between).

In certain embodiments, the flowable tissue matrix composition isirradiated to reduce bioburden, for example using 15-25 kGy E-beamirradiation.

Also disclosed herein, according to certain embodiments, are methods oftreatment, comprising implanting a flowable tissue matrix composition,as disclosed herein, into a tissue in need of repair, regeneration,healing, treatment, or alteration. In various embodiments, an implantedflowable tissue matrix composition provides a structural scaffold intowhich native cells from surrounding tissue can migrate and proliferate.In some embodiments, the implanted flowable tissue matrix compositionhas increased resistance to degradation, migration and/or resorption, ascompared to homogenized acellular tissue, while also retaining theability to flow into and mold to the shape of an implant site. In someembodiments, the implanted flowable tissue matrix composition reducesbleeding at an implant site (e.g., via the gelatin or gelatin gel morefully filling an implant site and blocking a source of bleeding).

In various embodiments, a flowable tissue matrix composition can beimplanted for cosmetic purposes, for example, in combination with abreast implant. In other embodiments, a flowable tissue matrixcomposition can be implanted following the removal of native tissue,such as a tumor. In some embodiments, implanting a flowable tissuematrix composition preserves the look or feel of native tissue after ithas been removed, as compared to the look or feel in the absence of animplanted flowable tissue matrix composition. In other embodiments, aflowable tissue matrix composition can be implanted following surgicalseparation of native tissues or in a wound or other void space thatoccurs through injury or disease. In some embodiments, implanting theflowable tissue matrix composition leads to faster healing, as comparedto healing in the absence of an implanted flowable tissue matrixcomposition.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cubes of acellular porcine dermis of different dimensions.

FIG. 2 shows gelatinized acellular porcine dermis (4.0% w/v).

FIG. 3 shows a gelatin gel comprising acellular porcine dermis (1.0%w/v, top syringe) and a cross-linked gelatin gel comprising acellularporcine dermis (0.5%, bottom syringe).

FIG. 4 shows a flowable tissue matrix composition comprising 30 g ofcubes of acellular porcine dermis (cube size 3.5 mm+/−0.2 mm) suspendedin 9 ml of a 1.0% gelatin gel comprising acellular porcine dermis.

FIG. 5 shows the relationship between cube size (measured in mm) and thesurface area to volume ratio (measured in mm²/mm³) for seven cubes thatcould be used in flowable tissue matrix compositions, and which wereproduced as described in example 1. The horizontal and vertical bars oneach data point indicate standard deviation.

FIG. 6A-B shows the effect of cube size on the resistance to collagenasedegradation for the flowable tissue matrix compositions producedaccording to examples 1-4. FIG. 6A shows the increase in free amineconcentration (in mM, normalized to a 1000 mg sample weight) foracellular porcine dermal cubes of different sizes (measured in mm) after18 hours in a collagenase solution. FIG. 6B shows the percentage oftissue remaining after incubating acellular porcine dermal cubes ofdifferent sizes (measured in mm) for 48 hours in a collagenase solution.

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings.

Disclosed herein are flowable tissue matrix compositions, comprisingsmall pieces of partially or completely decellularized tissue suspendedin a minimal volume of gel, comprising gelatinized tissue that has beenpartially or completely decellularized, or comprising a syntheticgelatin material. This two component system provides increased stabilityand resistance against migration and proteolytic degradation, while alsoretaining the flowable and moldable properties associated withparticulate acellular tissue. The flowable tissue matrices can be storedin hydrated form for extended periods of time and can be surgicallyimplanted as bulk soft tissue filler. For example, flowable tissuematrices can be implanted following the surgical removal of bulk softtissue, or as an implant for cosmetic purposes, or to fill a woundresulting from disease, trauma or surgery.

In some embodiments, a flowable tissue matrix composition is prepared byfirst preparing small pieces of acellular tissue by cutting or otherwiseprocessing partially or completely decellularized tissue into smallpieces. In certain embodiments, acellular or partially decellularizedtissue is gelatinized by incubating acellular or partiallydecellularized tissue in a solution containing a Lewis base such assodium carbonate, sodium citrate, and/or sodium acetate. In someembodiments, a gelatin gel is derived from gelatinized tissue by heatingthe tissue, for example to 40-60° C., and then cooling to roomtemperature. In certain embodiments, a gelatin gel can comprisebiocompatible synthetic material having a viscous consistency. In someembodiments, the gelatin gel can be cross-linked. In variousembodiments, cubes or other small pieces of acellular tissue aresuspended in the gelatinized tissue or gelatin gel. In certainembodiments, the amount of gelatinized tissue or gelatin gel isminimized, e.g., in order to minimize the volume of disrupted collagenin the composition. In some embodiments, the surface area to volumeratio of the small pieces of decellurized tissue is minimized (e.g., byreducing below a ratio of about 6 mm²/mm³), thereby increasing thecomposition's resistance to degradation, migration and/or resorption. Insome embodiments, the use of gelatin or gelatin gel also allows thecomposition to retain the flowable and moldable characteristics ofparticulate tissue.

The flowable tissue matrix compositions disclosed herein can be used, invarious embodiments, to repair, regenerate, heal, treat, and/or alter atissue in need thereof. For example, a flowable tissue matrixcomposition can be implanted to provide a biological or syntheticscaffold into which native cells from tissue surrounding thecompositions can migrate and proliferate, and which will resistdegradation or migration away from the site of implantation. Theflowable tissue matrices can be stored in hydrated form for extendedperiods of time and can be surgically implanted as a soft tissue fillerwithout the need to rehydrate the composition prior to use, therebyavoiding the risk of over-rehydrating and/or the delay associated withrehydration procedures. For example, the flowable tissue matrices can beimplanted following the surgical removal of bulk soft tissue, as animplant for cosmetic purposes, or to fill a wound or separated tissueresulting from disease, trauma or surgery. In addition, in certainembodiments, the gelatinized tissue or gelatin gel in a flowable tissuematrix composition can be used to help stop bleeding at an implant site.In some embodiments the flowable tissue matrix compositions can be usedto deliver enzymes, signaling molecules, or other factors to the tissuein need of repair, regeneration, or treatment, thereby promoting orenhancing the repopulation and/or revascularization of the implant withnative cells from surrounding tissue.

The materials and methods provided herein can be used to make abiocompatible, implantable composition. As used herein, a“biocompatible” composition is one that has the ability to support themigration and proliferation of native cells from surrounding tissue intothe composition following implantation. Biocompatible compositionssupport the native cellular activity necessary for tissue regeneration,repair, healing, or treatment and do not elicit a substantial immuneresponse that prevents such cellular activity. As used herein, a“substantial immune response” is one that prevents partial or completetissue regeneration, repair, healing, or treatment.

As used herein, the terms “native cells” and “native tissue” mean thecells or tissue present in the recipient organ or tissue prior toimplantation of a flowable tissue matrix composition, or the cells ortissue produced by the host animal after implantation.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose. To the extent publications and patentsor patent applications incorporated by reference contradict theinvention contained in the specification, the specification willsupersede any contradictory material.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. Also in this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”are not limiting. Any range described here will be understood to includethe endpoints and all values between the endpoints.

Flowable Tissue Matrices

In various embodiments, a flowable tissue matrix composition compriseshuman or animal tissue that has been at least partially decellularized.The tissue can be acellular, partially decellularized, and/ordecellularized tissue that has been repopulated with exogenous cells, solong as the tissue retains at least some of the extracellular matrixfound in the tissue prior to decellularizing.

In certain embodiments, a flowable tissue matrix composition can bederived from any human or animal tissue that is suitable for partial orcomplete decellularization and subsequent implantation. Exemplarytissues include, but are not limited to, bone, skin, dermis, intestine,urinary bladder, tendon, ligament, muscle, fascia, neurologic tissue,vascular tissue, vessel, liver, heart, lung, kidney, cartilage, and/orany other suitable tissue. In certain embodiments, a flowable tissuematrix composition can include a mammalian soft tissue. For example, incertain embodiments, a flowable tissue matrix composition can includepartially or completely decellularized mammalian dermis. As anotherexample, a flowable tissue matrix composition can comprise partially orcompletely decellularized mammalian small intestine submucosa, orpartially or completely decellularized mammalian lung or liver tissue. Aflowable tissue matrix composition can comprise tissue from one or more(e.g, 1, 2, 3, 4, 5, or more) different tissue sources. In certainembodiments, the decellularized tissue can come from human or non-humansources. Exemplary, suitable non-human tissue sources include, but arenot limited to, pigs, sheep, goats, cows, rabbits, monkeys, and/or othernon-human mammals. A flowable tissue matrix composition can comprisetissue from one or more (e.g, 1, 2, 3, 4, 5, or more) different animalsources.

In some embodiments, a flowable tissue matrix composition can be formedfrom ALLODERM® or STRATTICE™ (LIFECELL Corporation, Branchburg, N.J.),which are human and porcine acellular dermal matrices respectively.Alternatively, any other suitable acellular tissue matrices can be used.For example, a number of biological scaffold materials are described byBadylak et al., and the methods of the present disclosure can be used toproduce a stable three-dimensional acellular tissue matrix using any ofthose materials, or any other similar materials. Badylak et al.,“Extracellular Matrix as a Biological Scaffold Material: Structure andFunction,” Acta Biomaterialia (2008), doi:10.1016/j.actbio.2008.09.013,hereby incorporated by reference in its entirety.

In various embodiments, the extracellular scaffold within an acellularor partially decellularized tissue matrix may consist of collagen,elastin, or other fibers, as well as proteoglycans, polysaccharides andgrowth factors. The tissue matrix may retain some or all theextracellular matrix components that are found naturally in a tissueprior to decellularization, or various undesirable components may beremoved by chemical, enzymatic or genetic means. In general, theacellular matrix provides a structural network of fibers, proteoglycans,polysaccharides, and growth factors on which native tissue andvasculature can migrate, grow, and proliferate. The exact structuralcomponents of the extracellular matrix will depend on the tissueselected and the processes used to prepare the acellular or partiallydecellularized tissue.

In certain embodiments, a flowable tissue matrix composition lackscertain undesirable antigens. For example, certain animal tissuescontain alpha-galactose (α-gal) epitopes that are known to elicitreactions in humans. Therefore, flowable tissue matrix compositionsderived from various animal tissues can be produced or processed to lackcertain antigens, such as α-gal. In some embodiments, flowable tissuematrix compositions lack substantially all α-gal moieties. Eliminationof the α-gal epitopes may diminish the immune response against thecomposition. U. Galili et al., J. Biol. Chem. 263: 17755 (1988). Sincenon-primate mammals (e.g., pigs) produce α-gal epitopes,xenotransplantation of acellular tissue matrix material from thesemammals into primates may result, in some instances, in rejectionbecause of primate anti-gal binding to the α-gal epitopes on theacellular tissue matrix. The binding results in the destruction of theacellular tissue by complement fixation and by antibody-dependent cellcytotoxicity. U. Galili et al., Immunology Today 14: 480 (1993); M.Sandrin et al., Proc. Natl. Acad. Sci. USA 90: 11391 (1993); H. Good etal., Transplant. Proc. 24: 559 (1992); B. H. Collins et al., J. Immunol.154: 5500 (1995).

As described in detail below, in various embodiments, flowable tissuematrix compositions can be processed to remove antigens such as α-gal,e.g., by chemical or enzymatic treatment. Alternatively, in someembodiments, flowable tissue matrix compositions can be produced fromanimals that have been genetically modified to lack these epitopes.

Flowable tissue matrix compositions can be selected to provide a varietyof different biological and mechanical properties. For example, aflowable tissue matrix composition can be selected in order to provide ascaffold in which native cells from tissue surrounding an implantedcomposition can migrate and proliferate, thereby enhancing the speed oroverall level of repair, regeneration, healing, and/or treatment ofnative tissue. For example, an acellular tissue matrix, when implantedon or into fascia, may be selected to allow for regeneration of thefascia without excessive fibrosis or scar formation.

In certain embodiments, flowable tissue matrix compositions comprisinghuman or animal tissue are completely or substantially free of all cellsnormally present in the tissue from which the composition is derived. Asused herein, “substantially free of all cells” means that a flowabletissue matrix composition contains less than 20%, 10%, 5%, 1%, 0.1%,0.01%, 0.001%, or 0.0001% (or any percentage in between) of the cellsthat normally grow within the acellular matrix of the tissue prior todecellularization.

In some embodiments, flowable tissue matrix compositions can includeextracellular scaffolds that have been repopulated with viable cells.Various cell types can be used for repopulation, including stem cellssuch as embryonic stem cells, adult stem cells (e.g. mesenchymal stemcells), and/or neuronal cells. Any other viable cells can also be used.In some embodiments, the cells are mammalian cells. Such cells canpromote native tissue migration, proliferation, and/or vascularization.In various embodiments, the viable cells are applied to the acellulartissue matrix before or after implantation of a flowable tissue matrixcomposition.

In certain embodiments, flowable tissue matrix compositions comprise oneor more additional agents. In some embodiments, the additional agent(s)can comprise an anti-inflammatory agent, an analgesic, or any otherdesired therapeutic or beneficial agent. In certain embodiments, theadditional agent(s) can comprise, e.g., at least one added growth orsignaling factor (e.g., a cell growth factor, an angiogenic factor, adifferentiation factor, a cytokine, a hormone, and/or a chemokine).These additional agents can promote native tissue migration,proliferation, and/or vascularization. In some embodiments, the growthor signaling factor is encoded by a nucleic acid sequence containedwithin an expression vector. Preferably, the expression vector is in oneor more of the viable cells that can be added, optionally, to a flowabletissue matrix composition. As used herein, the term “expression vector”refers to any nucleic acid construct that is capable of being taken upby a cell, contains a nucleic acid sequence encoding a desired protein,and contains the other necessary nucleic acid sequences (e.g. promoters,enhancers, initiation and termination codons, etc.) to ensure at leastminimal expression of the desired protein by the cell.

In various embodiments, flowable tissue matrix compositions comprisesmall pieces of partially or completely decellularized tissue suspendedin decellularized tissue that has been gelatinized or processed into agelatinzed gel. In some embodiments, the small pieces of decellularizedtissue can have three dimensions (a length, a width, and a height) thatrange in size from about 1.0 mm to about 5.0 mm (e.g., about 1.0 mm, 1.5mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, or 5.0 mm, or anysize in between). In some embodiments, the pieces can have regularshapes (e.g., spheres, cubes, rhomboids) or irregular shapes, as long asthey generally have dimensions ranging from about 1.0 mm-5.0 mm.

In various embodiments, flowable tissue matrix compositions comprisesmall pieces of partially or completely decellularized tissue that aresuspended in a gelatin comprising homogenized acellular or partiallydecellularized tissue. In certain embodiments, the gelatin compriseshomogenized acellular or partially decellularized tissue suspended in anaqueous solution, wherein the homogenized tissue is present at aconcentration of about 0.1-10.0% w/v (dry tissue mass/total solutionvolume), e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, or 10.0% w/v (or any percentage in between). Insome embodiments, the aqueous solution can include a Lewis base (such assodium carbonate, sodium citrate, and/or sodium acetate), which is usedto expand and/or dissolve the acellular or partially decellularizedtissue within the gelatin, and a Lewis acid (such as HCl), which is usedto neutralize the Lewis base prior to combining the gelatin with thesmall pieces of partially or completely decellularized tissue.

In various embodiments, the small pieces of partially or completelydecellularized tissue are suspended in a gelatin gel comprisinggelatinized tissue that has been placed in a hydrating solution such asdistilled water, phosphate buffered saline (PBS), or any otherbiocompatible saline solution, heated, and then allowed to cool. Incertain embodiments, the decellularized tissue in the gelatin gel ispresent at about 0.1-10.0% w/v (dry tissue mass/total solution volume),e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0% w/v (or any percentage inbetween).

In certain embodiments, a gelatin gel comprises a biocompatiblesynthetic gel material having a viscous consistency, such as a hydrogel,starch gel, or other polysaccharide gel. In some embodiments, a gelatingel comprises one or more gelatinized tissues and one or more syntheticgel materials.

In various embodiments, the gelatin gel comprises homogenized acellularor partially decellularized tissue or a synthetic material that has beencross-linked. In certain embodiments, the cross-linked, decellularizedtissue or synthetic material in the gelatin gel is present at 0.1-10.0%w/v (dry mass/total solution volume), e.g., about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0,9.0, or 10.0% w/v (or any percentage in between).

In various embodiments, implanted flowable tissue matrix compositionshave increased resistance to degradation and/or resorption followingimplantation into a host tissue, as compared to an implanted homogenizedacellular tissue. In certain embodiments, the size of the pieces orfragments of acellular tissue within the flowable composition is aphysical parameter affecting the rheological properties of thecomposition, as well as a parameter regulating the biological responseupon implantation (for example, regulating the ability to resistdegradation, migration, and/or resorption). In this regard, the surfacearea to volume ratio of the small pieces of decellularized tissue withina flowable tissue matrix composition can alter the kinetics ofdegradation and remodeling, with larger pieces generally being moreresistant to degradation or migration. But, larger pieces are also oftenless flowable or malleable and additional gelatin or gelatin gel may berequired in order to adhere the larger pieces in an intact composition.Accordingly, the use of pieces of decellularized tissue having optimizeddimensions (e.g., in a range between about 1.0 mm and 5.0 mm) can enablethe flowable composition to exhibit the malleability of a homogenizedtissue, while avoiding rapid degradation and/or the need to use anincreased amount of gelatin or gelatin gel.

Accordingly, in some embodiments, the surface area to volume ratio ofeach piece of decellurized tissue in a flowable tissue matrixcomposition is minimized (e.g., by selecting or producing pieces oftissue having a ratio below about 6 mm²/mm³), such that the overallcomposition containing these small pieces can retain its moldablerheological properties using a minimal amount of gelatin or gelatin gelwhile increasing the composition's resistance to resorption, migration,and/or degradation (e.g., collagenase degradation) followingimplantation.

In certain embodiments, flowable tissue matrix compositions comprisesmall pieces of partially or completely decellularized tissue suspendedin a minimal volume of gelatinized tissue or gelatin gel. In certainembodiments, a “minimal volume” of gelatinized tissue or gelatin gel isthe amount that is sufficient to fill the space between the small piecesof decellularized tissue and/or which results in a flowable compositionthat retains structural integrity following implantation (e.g., wherethe small pieces of decellularized tissue remain clustered in closeproximity following implantation) and/or results in a flowablecomposition that resists degradation or migration followingimplantation. For example, a flowable tissue matrix composition cancomprise about 10-40 g (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 g, or anyamount in between) of decellularized tissue pieces having dimensions ofabout 1.0-5.0 mm (e.g., about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or5.0 mm, or any value in between) for every 1-15 ml of gelatinized tissueor gelatin gel (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 ml, or any volume in between). For example, a flowable tissuematrix composition can comprise about 30 g of decellularized tissuehaving dimensions of about 3.5 mm suspended in about 9 ml of about 1.0%gelatin gel. In another example, a flowable tissue matrix compositioncan comprise about 15 g of decellularized tissue having dimensions ofabout 2.2 mm suspended in about 5 ml of about 1.0% gelatin gel. In someembodiments, the surface area to volume ratio of each small piece ofdecellularized tissue is less than about 6 mm²/mm³ (e.g., less thanabout 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, 0.5, 0.4, or 0.3 mm²/mm³) (or anyvalue in between). In some embodiments, the small pieces of partially orcompletely decellularized tissue in a flowable tissue matrix compositionare selected such that a majority of the pieces have a surface area tovolume ratio less than about 6.0, 5.5, 5.0, or 4.5 mm²/mm³ (or any valuein between) and greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or 2.0 mm²/mm³ (or any value in between). As used herein,a “majority” indicates at least about 50% of the small pieces (e.g., atleast about 50, 55, 60, 65, 70 75, 80, 85, 90, 85, 99, or 99.9%) (or anypercentage in between).

Flowable tissue matrix compositions, as described above, may be packagedas frozen, freeze-dried, hydrated, and/or dehydrated products. Incertain embodiments, the packaged flowable tissue matrix compositionsare sterile. In certain embodiments, the flowable tissue matrixcompositions are provided in a kit, comprising a packaged flowabletissue matrix composition and instructions for preparing and/or usingthe flowable tissue matrix composition. In some embodiments, the kitcomprises a syringe or other device for delivering a flowable tissuematrix composition to a surgical implant site. In some embodiments, theflowable tissue matrix composition can be pre-loaded in hydrated form inthe delivery device to allow for delivery to an implant site withoutfirst requiring rehydrating or other processing steps.

Methods of Making Flowable Tissue Matrices

Disclosed herein are methods of making flowable tissue matrices. In someembodiments, the method comprises selecting a tissue containing anextracellular matrix; partially or completely decellularizing thetissue; processing the decellularized tissue to produce small pieces;gelatinizing some of the decellularized tissue; and combining the piecesof decellularized tissue with the gelatinized tissue.

In some embodiments, a flowable tissue matrix can be prepared from anytissue that is suitable for decellularization and subsequentimplantation. Exemplary tissues include, but are not limited to, bone,skin, dermis, intestine, urinary bladder, tendon, ligament, muscle,fascia, neurologic tissue, vascular tissue, vessel, liver, heart, lung,kidney, cartilage, and/or any other suitable tissue. In certainembodiments, the tissues can include a mammalian soft tissue. Forexample, in certain embodiments, the tissue can comprise mammaliandermis. In certain embodiments, the dermis can be separated fromsurrounding epidermis and/or other tissues, such as subcutaneous fat. Incertain embodiments, the tissue can comprise mammalian small intestinesubmucosa. In certain embodiments, the tissue can include human and/ornon-human sources. Exemplary, suitable non-human tissue sources include,but are not limited to, pigs, sheep, goats, cows, rabbits, monkeys,and/or other non-human mammals.

In some embodiments, a flowable tissue matrix is prepared by partiallyor completely decellularizing a donor tissue. Exemplary methods fordecellularizing tissue are disclosed in U.S. Pat. No. 6,933,326 and U.S.Patent Application 2010/0272782, which are hereby incorporated byreference in their entirety. In some embodiments, the decellularizedtissue provides a porous extracellular scaffold structure into whichcells from surrounding native tissue can migrate and proliferate afterimplantation into a host site. In certain exemplary embodiments, theacellular tissue comprises ALLODERM® or STRATTICE™, which are acellularhuman dermal products and porcine dermal products, respectively, and areavailable from LifeCell Corporation (Branchburg, N.J.).

In various embodiments, the general steps involved in the production ofan acellular or partially decellularized tissue matrix include providingtissue from a donor (e.g., a human cadaver or animal source) andremoving cells under conditions that preserve the biological andstructural functions of the extracellular matrix. In certainembodiments, the tissue can be washed to remove any residualcryoprotectants and/or other contaminants. Solutions used for washingcan be any physiologically-compatible solution. Examples of suitablewash solutions include distilled water, phosphate buffered saline (PBS),or any other biocompatible saline solution.

In certain embodiments, the washed tissue can be chemically treated tostabilize the tissue so as to avoid biochemical and/or structuraldegradation before, during, or after cell removal. In variousembodiments, the stabilizing solution arrests and prevents osmotic,hypoxic, autolytic, and/or proteolytic degradation; protects againstmicrobial contamination; and/or reduces mechanical damage that can occurduring decellularization of tissues that contain, for example, smoothmuscle components (e.g., blood vessels). The stabilizing solution maycontain an appropriate buffer, one or more antioxidants, one or moreoncotic agents, one or more antibiotics, one or more proteaseinhibitors, and/or one or more smooth muscle relaxants.

In various embodiments, the tissue can be placed in a decellularizationsolution to remove viable and non-viable cells (e.g., epithelial cells,endothelial cells, smooth muscle cells, and fibroblasts, etc.) from theextracellular matrix without damaging the biological and/or structuralintegrity of the extracellular matrix. The decellularization solutionmay contain an appropriate buffer, salt, an antibiotic, one or moredetergents (e.g., TRITON X-100™ sodium dodecyl sulfate, sodiumdeoxycholate, polyoxyethylene (20) sorbitan mono-oleate, etc.), one ormore agents to prevent cross-linking, one or more protease inhibitors,and/or one or more enzymes. In some embodiments, the decellularizationsolution comprises 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, or 5.0% (or any percentage in between) of TRITONX-100™ and, optionally, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM,45 mM, or 50 mM EDTA (ethylenediaminetetraacetic acid) (or anyconcentration in between). In certain embodiments, the decellularizationsolution comprises 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, or 5.0% (or any percentage in between) of sodiumdeoxycholate and, optionally, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM,8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or 20 mM HEPESbuffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) containing10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM EDTA(or any concentrations in between). In some embodiments, the tissue isincubated in the decellularization solution at 20, 21, 22, 23, 24, 25,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 degrees Celsius(or any temperature in between), and optionally, gentle shaking isapplied at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,or 150 rpm (or any rpm in between). The incubation can be for 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36, 48, or 96 hours (or anytime in between). The length of time of exposure to thedecellularization solution, or the concentration of detergent and/orother decellularizing agents can be adjusted in order to partially ormore fully decellularize the tissue. In certain embodiments, additionaldetergents may be used to remove cells from the tissue sample. Forexample, in some embodiments, sodium deoxycholate and TRITON X-100™ areused to decellularize and separate other undesired tissue componentsfrom the extracellular tissue matrix.

In certain embodiments, the decellularized tissue can be placed in asolution containing calcium hydroxide. In some embodiments, the calciumhydroxide is at a concentration of about 0.05%-1.0% (w/v) calciumhydroxide (e.g., about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45,0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% w/v) (or any percentage in between). Insome embodiments, the tissue is placed in the calcium hydroxide solutionat about 20-40° C. (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40° C.) (or any temperaturein between). In some embodiments, the tissue is placed in the calciumhydroxide solution for about 1-5 days (e.g., about 1, 2, 3, 4, or 5days, or any time period in between). In some embodiments, the calciumhydroxide solution serves to dissolve undesired tissue components. Forexample, where the tissue is dermal tissue, the calcium hydroxidesolution can dissolve epidermis and enable the manual removal of hairfollicles. In certain embodiments, after calcium hydroxide treatment,the calcium hydroxide can be neutralized, for example using acetic acid.

In some embodiments, after decellularization, the tissue sample iswashed thoroughly. Any physiologically compatible solutions can be usedfor washing. Examples of suitable wash solutions include distilledwater, phosphate buffered saline (PBS), or any other biocompatiblesaline solution. In certain embodiments, e.g., when xenogenic orallogenic material is used, the decellularized tissue is then treatedovernight at room temperature with a deoxyribonuclease (DNase) solution.In some embodiments, the tissue sample is treated with a DNase solutionprepared in DNase buffer (20 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM CaCl₂ and 20mM MgCl₂). Optionally, an antibiotic solution (e.g., Gentamicin) may beadded to the DNase solution. Any suitable DNase buffer can be used, aslong as the buffer provides for suitable DNase activity.

While an acellular or partially decellularized tissue matrix may bederived from tissue from one or more donor animals of the same speciesas the intended recipient animal, this is not necessarily the case.Thus, for example, an acellular tissue matrix may be derived fromporcine tissue and implanted in a human patient. Species that can serveas donors and/or recipients of acellular tissue matrices include,without limitation, mammals, such as humans, nonhuman primates (e.g.,monkeys, baboons, or chimpanzees), pigs, cows, horses, goats, sheep,dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice.

In certain embodiments, decellularized tissue can be treated with one ormore enzymes to remove undesirable antigens, e.g., an antigen notnormally expressed by the recipient animal and thus likely to lead to animmune response and/or rejection of the implanted flowable tissue matrixcomposition. For example, in certain embodiments, decellularized tissuecan be treated with alpha-galactosidase to remove alpha-galactose(α-gal) moieties. In some embodiments, to enzymatically remove α-galepitopes, after washing tissue thoroughly with saline, the tissue may besubjected to one or more enzymatic treatments to remove α-gal antigens,if present in the sample. In certain embodiments, the tissue may betreated with an α-galactosidase enzyme to eliminate α-gal epitopes. Inone embodiment, the tissue is treated with α-galactosidase at aconcentration of 0.2 U/ml prepared in 100 mM phosphate buffered salineat pH 6.0. In other embodiments, the concentration of α-galactosidase isreduced to 0.1 U/ml or increased to 0.3, 0.4, or 0.5 U/ml (or any valuein between). In other embodiments, any suitable enzyme concentration andbuffer can be used, as long as sufficient antigen removal is achieved.In addition, certain exemplary methods of processing tissues to reduceor remove alpha-1,3-galactose moieties are described in Xu et al.,Tissue Engineering, Vol. 15, 1-13 (2009), which is hereby incorporatedby reference in its entirety.

In certain embodiments, animals that have been genetically modified tolack one or more antigenic epitopes may be selected as the tissue sourcefor a flowable tissue matrix composition. For example, animals (e.g.,pigs) that have been genetically engineered to lack the terminalα-galactose moiety can be selected as the tissue source. Fordescriptions of appropriate animals and methods of producing transgenicanimals for xenotransplantation, see U.S. patent application Ser. No.10/896,594 and U.S. Pat. No. 6,166,288, which are hereby incorporated byreference in their entirety.

In some embodiments, the decellularized tissue can be treated to reducebioburden (i.e., to reduce the number of microorganisms growing on thetissue). In some embodiments, the tissue is treated such that it lackssubstantially all bioburden (i.e., the tissue is aseptic or sterile).Suitable bioburden reduction methods are known to one of skill in theart, and may include exposing the tissue to radiation. Irradiation mayreduce or substantially eliminate bioburden. In some embodiments, anabsorbed dose of 15-17 kGy of e-beam radiation is delivered in order toreduce or substantially eliminate bioburden. In various embodiments, aflowable tissue matrix composition is exposed to between about 5 Gy and50 kGy of radiation. Suitable forms of radiation can include gammaradiation, e-beam radiation, and X-ray radiation. Other irradiationmethods are described in U.S. Application 2010/0272782, the disclosureof which is hereby incorporated by reference in its entirety.

In certain embodiments, after decellularization, viable cells mayoptionally be seeded in the extracellular matrix. In some embodiments,viable cells may be added to the matrix by standard in vitro cellco-culturing techniques prior to transplantation, or by in vivorepopulation following transplantation. In vivo repopulation can be bythe migration of native cells from surrounding tissue into the matrix orby infusing or injecting viable cells obtained from the recipient orfrom another donor into the matrix in situ. Various cell types can beused, including stem cells such as embryonic stem cells and/or adultstem cells (e.g. mesenchymal stem cells). Any other viable cells canalso be used. In some embodiments, the cells are mammalian cells. Incertain embodiments, the cells are histocompatible with the subject inwhich they are implanted. Such cells can promote native tissuemigration, proliferation, and/or vascularization. In variousembodiments, the cells can be directly applied to the matrix of adecellularized tissue just before or after implantation.

In certain embodiments, one or more additional agents can be added tothe extracellular matrix of a decellularized tissue. In someembodiments, the additional agent can comprise an anti-inflammatoryagent, an analgesic, or any other desired therapeutic or beneficialagent. In certain embodiments, the additional agent can comprise atleast one added growth or signaling factor (e.g., a cell growth factor,an angiogenic factor, a differentiation factor, a cytokine, a hormone,and/or a chemokine). In some embodiments, these additional agents canpromote native tissue migration, proliferation, and/or vascularizationwithin the extracellular matrix. In some embodiments, the growth orsignaling factor is encoded by a nucleic acid sequence contained withinan expression vector. Preferably, the expression vector is in one ormore of the viable cells that can be included, optionally, in theextracellular matrix of the decellularized tissue. As used herein, theterm “expression vector” refers to any nucleic acid construct that iscapable of being taken up by a cell, contains a nucleic acid sequenceencoding a desired protein, and contains the other necessary nucleicacid sequences (e.g. promoters, enhancers, termination codon, etc.) toensure at least minimal expression of the desired protein by the cell.

In various embodiments, the decellularized tissue can be processed intosmall pieces. In some embodiments, the small pieces are selected to havedimensions that minimize their surface area to volume ratio (e.g., asurface area to volume ratio of less than or equal to about 6 mm²/mm³).For example, the decellularized tissue can be cut, e.g., using a scalpelor razor, to form small cubes. In some embodiments, the small pieces canhave three dimensions (a length, a width, and a height) that each rangein size from about 1.0 mm to about 5.0 mm (e.g., about 1.0 mm, 1.5 mm,2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, or 5.0 mm, or any sizein between). In some embodiments, the pieces can have regular shapes(e.g., spheres, cubes, rhomboids) or irregular shapes, as long as theygenerally have dimensions ranging from about 1.0 mm-5.0 mm.

In various embodiments, partially or completely decellularized tissue isgelatinized. In some embodiments, the decellularized tissue is firstsuspended in an aqueous solution containing a Lewis base, such as sodiumcarbonate, sodium citrate, and/or sodium acetate. In some embodiments,the Lewis base is present in the solution at a concentration of about10-30 mM (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 mM, or any concentration inbetween). In certain embodiments, the decellularized tissue is presentin the aqueous solution at about 0.1-10.0% w/v (dry tissue mass/totalsolution volume), e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0% w/v (or any percentage inbetween). In various embodiments, the decellularized tissue is incubatedin the basic solution, with or without agitation, at a temperature ofabout 40-75° C. (e.g, about 40, 45, 50, 55, 60, 65, 70, or 75° C., orany temperature in between) for about 10-48 hours (e.g., about 10, 15,20, 24, 36, or 48 hours, or any time period in between). In someembodiments, after incubation, the Lewis base in the suspension isneutralized, for example using HCl at a concentration of about 0.05-0.5M (e.g., about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5M, or any concentration in between). In various embodiments, thedecellularized tissue suspension is homogenized to form a gelatin. Insome embodiments, decellularized tissue is homogenized before incubationin the basic solution, while in other embodiments it is homogenizedduring or after incubation.

In some embodiments, the gelatinized tissue can be suspended in ahydrating solution. Suitable hydrating solutions include distilledwater, phosphate buffered saline (PBS), or any other biocompatiblesaline solution. In some embodiments, the biocompatible saline solutionis at a concentration of about 0.1-10% saline (e.g., about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0%, or any percentage in between).In certain embodiments, the saline suspension is heated to about 40-60°C. (e.g., about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, or 60° C., or any temperature in between) andthen allowed to cool to room temperature, forming a viscous gelatin gel.

In various embodiments, a gelatin gel can be prepared by selecting abiocompatible synthetic gel material having a viscous consistency, suchas a hydrogel, starch gel, or other polysaccharide gel. In someembodiments, a gelatin gel is prepared by combining one or moregelatinized tissues with one or more synthetic gel materials.

In certain embodiments, the decellularized tissue or synthetic materialin the gelatin gel is present at about 0.1-10.0% w/v (dry mass/totalsolution volume), e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0% w/v (orany percentage in between).

In some embodiments, a cross-linked gelatin gel can be prepared byadding a cross-linking agent (such as glutaraldehyde, genipin, and/orthe reversible cross-linking agent 1, 2, 3, 4, 6-pentagalloyl glucose(PGG)) at a concentration of about 0.01-2.0% (w/v) (e.g., about 0.01,0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,or 2.0%, or any percentage in between). In certain embodiments, thecross-linking reaction is allowed to proceed at approximately roomtemperature (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28° C.,or any temperature in between) for 12-60 hours (e.g., 12, 15, 20, 24,36, 48, or 60 hours, or any time period in between). In certainembodiments, the cross-linked gelatin gel is at a concentration of about0.1-10.0% w/v (dry mass/total solution volume), e.g., about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0,7.0, 8.0, 9.0, or 10.0% w/v (or any percentage in between).

In various embodiments, an implanted flowable tissue matrix compositionis prepared such that it has increased resistance to degradation,migration, and/or resorption following implantation into a host tissue,as compared to an implanted homogenized acellular tissue. In certainembodiments, the size of the pieces or fragments of acellular tissuewithin a flowable composition is a physical parameter affecting therheological properties, and is also a factor for regulating thebiological response upon implantation, for example the ability to resistdegradation, migration, and/or resorption. In this regard, in certainembodiments, changes in the surface area to volume ratio of the piecesof decellularized tissue in the flowable composition can alter thekinetics of degradation and remodeling.

Accordingly, in various embodiments, the surface area to volume ratiofor a majority of the pieces of decellularized tissue in a flowabletissue matrix composition is minimized (e.g., by reducing the ratiobelow about 6 mm²/mm³), such that the composition containing these smallpieces exhibits moldable rheological properties while increasingresistance to resorption, migration, and/or degradation (e.g.,collagenase degradation) following implantation. In some embodiments,the optimized small pieces of decellularized tissue can be combined withgelatinized tissue or gelatin gel. In certain embodiments, the volume ofgelatinized tissue or gelatin gel is minimized. In some embodiments, a“minimal volume” of gelatinized tissue or gelatin gel is the amountsufficient to fill the space between the small pieces of decellularizedtissue and to allow for an effective flowable composition. For example,about 10-40 g (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 g, or any amount inbetween) of decellularized tissue pieces having dimensions of about1.0-5.0 mm (e.g., about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0mm, or any value in between) can be combined with every 1-15 ml ofgelatinized tissue or gelatin gel (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 ml, or any value in between). For example,about 30 g of decellularized tissue having dimensions of about 3.5 mmcan be combined with about 9 ml of about 1% gelatin gel to form aflowable tissue matrix composition. In another example, about 15 g ofdecellularized tissue having dimensions of about 2.2 mm can be combinedwith about 5 ml of about 1% gelatin gel to form a flowable tissue matrixcomposition.

In certain embodiments, a flowable tissue matrix composition cancomprise, by volume, about 70 to 80% pieces of acellular tissue (e.g.,about 70, 72, 74, 76, 78, or 80% or any percentage in between), whilethe remaining approximately 20 to 30% of the composition (e.g., about20, 22, 24, 26, 28 or 30% or any percentage in between) can comprisegelatinized tissue or gelatin gel. In some embodiments, a flowabletissue matrix composition can comprise, by mass, about 90 to 98% piecesof acellular tissue (e.g., about 90, 92, 94, 96, or 98% or anypercentage in between), while the remaining approximately 2 to 8% of thecomposition (e.g., about 2, 4, 6, or 8% or any percentage in between)can comprise gelatinized tissue or gelatin gel. In some embodiments, thesurface area to volume ratio of the small pieces of decellularizedtissue in a flowable tissue matrix composition is less than about 6mm²/mm³ (e.g., less than about 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, 0.5, 0.4,0.3, 0.2, or 0.1 mm²/mm³) (or any value in between).

Methods of Use

Disclosed herein are methods of using the flowable tissue matrixcompositions described above.

In various embodiments, the flowable tissue matrix compositions can beimplanted into a host tissue in need of repair, regeneration, treatment,and/or enhancement. The extracellular matrix provided by the partiallyor completely decellularized tissue in the compositions provides ascaffold into which native cells from surrounding tissue can migrate andproliferate. Accordingly, in certain embodiments, the extracellularscaffold in a flowable tissue matrix composition can enhance and/orpromote tissue treatment, repair, and/or regeneration. Furthermore, asdiscussed in more detail below, flowable tissue matrix compositions canbe used, in certain embodiments, to mold to the shape of an implant sitewhile resisting degradation and/or resorption.

It is known that small particles of acellular tissue (e.g., those havingdimensions of less than about 25 microns) are prone to migrate away froman implant site and are more susceptible to degradation followingimplantation. Furthermore, small particles of acellular tissue are noteasily stored in the hydrated state, due to hydrolytic activity andnatural phase separation. Thus, such particles are often storeddehydrated, thereby requiring lengthy rehydration and the possibility ofover-rehydration prior to surgical use.

Accordingly, in various embodiments, the flowable tissue matrixcompositions disclosed herein can overcome the problems associated withthe storage and use of small particulate acellular tissue by providingan implantable material that can be stored in hydrated form and whichprovides increased resistance to degradation and/or resorption followingimplantation into a host tissue, as compared to an implanted homogenizedacellular tissue comprising small particles of less than 25 microns insize, while preserving the desirable moldable properties of particulatetissue. In some embodiments, the surface area to volume ratio of thesmall pieces of decellularized tissue in a flowable tissue matrixcomposition is minimized (e.g., by reducing the ratio below about 6mm²/mm³), for example by using small pieces of decellularized tissuehaving dimensions of between about 1.0 mm and 5.0 mm. In someembodiments, these small pieces of decellularized tissue are suspendedin a minimal amount of gelatinized tissue or gelatin gel (e.g., anamount that minimally fills the spaces between the pieces of tissue),such that the flowable tissue matrix composition retains its moldableproperties while increasing its resistance to resorption, migration,and/or degradation (e.g. collagenase degradation) followingimplantation. In some embodiments, a flowable tissue matrix compositionhas increased resistance to collagenase degradation, which can bemeasured, for example, by determining whether at least about 20% (e.g.,at least about 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, or anypercentage in between) of the composition remains after 48 hours of invitro exposure to about 5 units/mL of Type I collagenase, or after 48hours of in vivo exposure to an implant site. In certain embodiments, animplanted flowable tissue matrix composition can conform to the contoursof an implant site, thereby completely filling the implant site and/ormolding to provide support for a desired structure or shape for theimplant.

In various embodiments, an implanted flowable tissue matrix compositionprovides a biocompatible scaffold that supports the native tissuemigration, proliferation, and/or revascularization necessary for tissueregeneration, repair, healing, and/or treatment, and does not elicit asubstantial immune response that prevents such activity. As used herein,a “substantial immune response” is one that prevents partial or completetissue regeneration, repair, healing, and/or treatment. In certainembodiments, an implanted flowable tissue matrix composition lackscertain undesirable antigens in order to avoid inducing an immuneresponse. For example, in some embodiments, an implanted flowable tissuematrix composition lacks substantially all α-gal moieties that are knownto elicit reactions in humans.

In certain embodiments, the flowable tissue matrix compositions that areimplanted in a patient comprise human and/or animal tissue that iscompletely or substantially free of all cells normally present in thetissue from which the flowable tissue matrix composition is derived. Asused herein, “substantially free of all cells” means that the flowabletissue matrix composition contains less than 20%, 10%, 5%, 1%, 0.1%,0.01%, 0.001%, or 0.0001% (or any percentage in between) of the cellsthat normally grow within the acellular matrix of the tissue prior todecellularization.

In some embodiments, the implanted flowable tissue matrix compositionscan include an extracellular scaffold that has been repopulated withviable cells. Various cell types can be used for repopulation, includingstem cells such as embryonic stem cells, adult stem cells (e.g.mesenchymal stem cells), and/or neuronal cells. Any other viable cellscan also be used. In some embodiments, the cells are mammalian cells.Such cells can promote native tissue migration, proliferation, and/orrevascularization. In various embodiments, the viable cells are appliedto the extracellular scaffold of a flowable tissue matrix compositionbefore or after implantation.

In certain embodiments, an implanted flowable tissue matrix compositionfurther comprises one or more additional agents. In some embodiments,the additional agent can comprise an anti-inflammatory agent, ananalgesic, or any other desired therapeutic or beneficial agent thatpromotes tissue repair, regeneration, and/or treatment followingimplantation. In certain embodiments, the additional agent can comprise,e.g., at least one added growth or signaling factor (e.g., a cell growthfactor, an angiogenic factor, a differentiation factor, a cytokine, ahormone, and/or a chemokine). These additional agents can promote nativetissue migration, proliferation, and/or vascularization.

Flowable tissue matrix compositions can be implanted in a patient aspart of any medical procedure in which tissue repair, regeneration, ortreatment is desired. For example, flowable tissue matrix compositionscan be implanted following the creation of space between tissue planesas a result of disease, trauma, or surgical intervention. In someembodiments, the composition can be implanted into a space betweenseparated tissue planes and molded to fill the anatomical shape of theimplant site. In various embodiments, the implanted composition canprovide a scaffold for native tissue migration, proliferation, andrevascularization. In certain embodiments, the gelatinized tissue orgelatin gel in a flowable tissue matrix composition can also be used tohelp reduce or prevent bleeding at the site of implantation.

In another example, flowable tissue matrix compositions can be used astissue fillers by implanting them following the removal of bulk softtissue from a patient, e.g., tumor removal. It has been shown that aftertumor removal, tissue re-growth is generally poor, especially as to thesubcutaneous tissue layers. Generally, a layer of skin will regrow aftertumor removal, but the underlying tissue remains unregenerated. Thus, invarious embodiments, flowable tissue matrix compositions can be used asimplants to replace bulk soft tissue after tumor removal. In certainembodiments, such implants serve as tissue fillers that can be molded tothe shape of the implant site while resisting degradation, migration,and/or resorption. In certain embodiments, where the bulk tissue that isremoved is near or includes the skin, implantation of a flowable tissuematrix composition can provide the implant site with a more natural lookand/or feel after tumor removal. In various embodiments, the implantedcomposition can also provide a scaffold for native tissue migration,proliferation, and/or revascularization.

In yet another example, flowable tissue matrix compositions can be usedfor aesthetic purposes, e.g., as implants or in conjunction withtraditional implants. For example, flowable tissue matrix compositionscan be used to support traditional breast implants, e.g., for use inbreast augmentation and/or reconstruction. For example, a flowabletissue matrix composition can be placed around a breast implant and usedto fill the space between the implant and surrounding native tissue,thereby providing a smoother contour and/or more natural look and feelfor the implant. At the same time, in certain embodiments, the implantedflowable tissue matrix composition can provide a scaffold into whichcells from native tissue surrounding the breast implant can grow andproliferate, thereby more firmly securing the breast implant in placeand/or reducing the amount of undesirable fibrosis that develops aroundthe implant. In other embodiments, a flowable tissue matrix compositioncan be used independently as an implant, for example as a collagenimplant to increase tissue volume (e.g., lip injections).

EXAMPLES

The following examples serve to illustrate, and in no way limit, thepresent disclosure.

Example 1: Preparation of Acellular Porcine Dermal ExtracellularMatrices

Raw porcine hides were obtained from an abattoir. To prevent thedenaturation of the dermal extracellular matrix, the hides were chilledin a refrigerator at 2° C. to 10° C. Subcutaneous fat was mechanicallyremoved from the hides, and skin material was briefly soaked and cleanedin 0.5% (w/v) Triton X-100 solution. Cleaned skin was 3 to 4 mm thickand was trimmed to smaller pieces of about ˜8 cm×10 cm.

Skin pieces were then soaked with agitation at room temperature (22° C.to 25° C.) for two days in 0.2% (w/v) calcium hydroxide. The solution totissue ratio was 500 mL of solution per 100 g of tissue. Epidermis wasdissolved during soaking, and after a 30 minute rinse with distilledwater, hairs were plucked. After soaking dermal sheets with agitation in0.2% (w/v) calcium hydroxide for another 3 days, hair follicles werepressed out. Calcium hydroxide treated dermal sheets were washed withdistilled water twice, and neutralized with acetic acid to a pH of 7.5.Calcium residue was rinsed off with agitation in distilled water for 8hours.

Then, dermal sheets were soaked with agitation in a 2.0% (w/v) sodiumdeoxycholate (SDC) solution dissolved in 10 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 8.0)containing 10 mM EDTA (ethylenediaminetetraacetic acid). After SDCtreatment at 37° C. for 40 hours, dermal sheets were washed inDulbecco's phosphate buffered saline (PBS) containing 5 mM EDTA (pH 7.3)overnight (˜18 hours). The wash solution was changed three times duringthis washing step. Processed dermal sheets were cut into small cubes atsizes ranging from 1 mm to 5 mm (see FIG. 1 for some examples of suchcubes).

Example 2: Preparation of Gelatinized Acellular Porcine Dermis

Cubes of porcine dermal acellular tissue (dECM) were suspended in a 20mM sodium carbonate solution (sterile-filtrated) at 100-mL solution per4 g dry dECM mass and incubated with agitation at 55° C. to 65° C. overnight (˜18 hours). The gelatinized tissue suspension was neutralizedwith 0.1 M HCl before being homogenized into a dECM gel (FIG. 2).Alternatively, dECM cubes were homogenized first in sodium carbonatesolution before incubation at 55° C. to 65° C.

Example 3: Preparation of a Purified Gelatin Gel

Purified porcine skin gelatin (Sigma-Aldrich, St. Louis, Miss.) wasdissolved in 0.9% saline at 50° C. Upon cooling to room temperature, thegelatin suspension became a highly viscous gel (FIG. 3). A cross-linkedversion of gelatin gel was made with a reversible cross-linker, PGG(1,2,3,4,6-pentagalloyl glucose). An equal volume of 1.0% gelatin geland 0.1% (w/v) PGG solution was mixed. The cross-linking reactionproceeded at room temperature (22° C. to 25° C.) for 48 hours.

Example 4: Preparation of a Flowable dECM Composition

Flowable dECM compositions were prepared by mixing dECM cubes (describedin example 1) with gelatinzed dECM (described in example 2) or gelatingel (described in example 3). The gel volume was kept to the minimumrequired to fill the void space between dECM cubes and to achieve astable flowable composition. FIG. 4 shows a stable flowable compositionprepared with 30 g of porcine dECM cubes (3.5±0.2 mm) in 9 mL of a 1.0%gelatin gel. A similar composition was made with 15 g of smaller dECMcubes (2.2±0.1 mm) in 5 mL of a 1.0% gelatin gel.

Example 5: Measurement of the Surface Area to Volume Ratio

To calculate the volume of a dECM cube, its mass was weighed, and thedECM cube's volume was calculated using its specific density, which wasmeasured at 1.06 g/cm³ by the displacement method. The surface area ofthe dECM cube was calculated from the dimensions of its faces. FIG. 5shows the surface area to volume relationship for seven dECM cubes ofdifferent sizes. The surface area to volume ratio increases rapidly whenthe dECM cube has dimensions below 1.0 mm.

Example 6: Resistance to Collagenase Degradation

The resistance of dECM cubes of different sizes to collagenasedegradation was tested in vitro using type I collagenase. Samples ofdECM cubes (697 mg±52 mg) were placed into 30 mL of 10 mM Tris-HClbuffer (pH 7.5) containing 2 mM calcium chloride. Type I collagenase wasadded to a final activity of 5 units/ml, and samples were incubated withgentle agitation at 37° C. The increase in free amine content in thedegradation solution was determined after 18 hours incubation and theamount of tissue remaining was recorded after 48 hours incubation. After18 hours, a 20-μL aliquot of degradation solution was mixed with 500 μLof 100 mM sodium bicarbonate solution containing 0.05% picryl sulfonicacid (PSA), and reacted at 37° C. for 2 hours. Then 300 μL of 2 M HCland 2.4 mL of distilled water were added into each sample. The increasein free amine concentration due to protein degradation was determinedspectrophotometrically (345 nm) using glycine as a standard. FIG. 6A-Bshow the effect of dECM cube size on the resistance to collagenasedegradation. As dECM cube size decreased, the susceptibility tocollagenase degradation increased.

The preceding examples are intended to illustrate and in no way limitthe present disclosure. Other embodiments of the disclosed devices andmethods will be apparent to those skilled in the art from considerationof the specification and practice of the devices and methods disclosedherein.

What is claimed is:
 1. A flowable tissue matrix composition, comprising small pieces of partially or completely decellularized tissue, wherein a majority of the small pieces of partially or completely decellularized tissue have surface area to volume ratio of less than about 6 mm²/mm³ and wherein the small pieces of partially or completely decellularized tissue are suspended in a gelatin gel comprising less than about 20% of the composition on a volume/volume (v/v) basis.
 2. The composition of claim 1, wherein the small pieces of partially or completely decellularized tissue in the composition have an increased resistance to degradation or resorption as measured using a type I collagenase digestion assay, as compared to a homogenized acellular tissue, while also retaining the ability to flow into and mold to the shape of an implant site.
 3. The composition of claim 1, wherein the small pieces of partially or completely decellularized tissue have a length, a width, and a height ranging from about 1.0 mm to about 5.0 mm.
 4. The composition of claim 3, wherein the length, width, and height of the majority of the small pieces of partially or completely decellularized tissue ranges from about 2.0 mm to 4.0 mm.
 5. The composition of claim 1, wherein the majority of the small pieces of partially or completely decellularized tissue in the composition have a surface area to volume ratio of greater than about 1.5 mm²/mm³ and less than about 6 mm²/mm³.
 6. The composition of claim 1, wherein the majority of the small pieces of partially or completely decellularized tissue in the composition have a surface area to volume ratio of less than about 4.5 mm²/mm³ and greater than about 2.0 mm²/mm³.
 7. The composition of claim 1, wherein the gelatin gel is crosslinked.
 8. The composition of claim 1, wherein the small pieces of partially or completely decellularized tissue are derived from an animal selected from a group consisting of human, nonhuman primate, pig, cow, horse, goat, sheep, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, and mouse.
 9. The composition of claim 1, wherein the small pieces of partially or completely decellularized tissue are derived from a tissue selected from a group consisting of bone, skin, dermis, intestine, vascular, urinary bladder, tendon, ligament, muscle, fascia, neurologic tissue, vessel, liver, heart, lung, kidney, and cartilage tissue.
 10. The composition of claim 1, wherein the flowable tissue matrix composition lacks alpha-galactose moieties.
 11. The composition of claim 1, further comprising one or more viable cells.
 12. The composition of claim 11, wherein the one or more viable cells are mammalian cells.
 13. The composition of claim 11, wherein the one or more viable cells are stem cells.
 14. The composition of claim 1, further comprising at least one additional factor selected from a group consisting of an anti-inflammatory agent, an analgesic, a cell growth factor, an angiogenic factor, a differentiation factor, a cytokine, a hormone, and a chemokine.
 15. The composition of claim 14, wherein the at least one additional factor is encoded by a nucleic acid sequence contained within an expression vector.
 16. The composition of claim 15, wherein the expression vector is contained within one or more viable cells.
 17. The composition of claim 1, wherein the flowable tissue matrix composition has been subjected to a sterilization process.
 18. The composition of claim 1, wherein the gelatin gel comprises a gelatinized tissue that has been heated to at least about 50° C. and then allowed to cool.
 19. The composition of claim 18, wherein the gelatinized tissue comprises homogenized acellular or partially decellularized tissue in an aqueous solution at a concentration of about 0.1-10.0% weight/volume (w/v).
 20. The composition of claim 1, wherein the gelatin gel comprises at least one of a hydrogel or a starch gel. 